Obesity-associated gene TMEM18 has a role in the central control of appetite and body weight regulation.

An intergenic region of human chromosome 2 (2p25.3) harbors genetic variants which are among those most strongly and reproducibly associated with obesity. The gene closest to these variants is TMEM18, although the molecular mechanisms mediating these effects remain entirely unknown. Tmem18 expression in the murine hypothalamic paraventricular nucleus (PVN) was altered by changes in nutritional state. Germline loss of Tmem18 in mice resulted in increased body weight, which was exacerbated by high fat diet and driven by increased food intake. Selective overexpression of Tmem18 in the PVN of wild-type mice reduced food intake and also increased energy expenditure. We provide evidence that TMEM18 has four, not three, transmembrane domains and that it physically interacts with key components of the nuclear pore complex. Our data support the hypothesis that TMEM18 itself, acting within the central nervous system, is a plausible mediator of the impact of adjacent genetic variation on human adiposity.

Analysis of naturally occurring mutations in the human lipodystrophy protein seipin reveals multiple potential pathogenic mechanisms.

AIMS/HYPOTHESIS: In humans, disruption of the gene BSCL2, encoding the protein seipin, causes congenital generalised lipodystrophy (CGL) with severe insulin resistance and dyslipidaemia. While the causative gene has been known for over a decade, the molecular functions of seipin are only now being uncovered. Most pathogenic mutations in BSCL2 represent substantial disruptions including significant deletions and frameshifts. However, several more subtle mutations have been reported that cause premature stop codons or single amino acid substitutions. Here we have examined these mutant forms of seipin to gain insight into how they may cause CGL. METHODS: We generated constructs expressing mutant seipin proteins and determined their expression and localisation. We also assessed their capacity to recruit the key adipogenic phosphatidic acid phosphatase lipin 1, a recently identified molecular role of seipin in developing adipocytes. Finally, we used atomic force microscopy to define the oligomeric structure of seipin and to determine whether this is affected by the mutations. RESULTS: We show that the R275X mutant of seipin is not expressed in pre-adipocytes. While the other premature stop mutant forms fail to bind lipin 1 appropriately, the point mutants T78A, L91P and A212P all retain this capacity. We demonstrate that wild-type human seipin forms oligomers of 12 subunits in a circular configuration but that the L91P and A212P mutants of seipin do not. CONCLUSIONS/INTERPRETATION: Our study represents the most comprehensive analysis so far of mutants of seipin causing lipodystrophy and reveals several different molecular mechanisms by which these mutations may cause disease.

HOXC10 Suppresses Browning to Maintain White Adipocyte Identity.

Promoting beige adipocyte development within white adipose tissue (WAT) is a potential therapeutic approach to staunch the current obesity epidemic. Previously, we identified homeobox-containing transcription factor HOXC10 as a suppressor of browning in subcutaneous WAT. Here, we provide evidence for the physiological role of HOXC10 in regulating WAT thermogenesis. Analysis of an adipose-specific HOXC10 knockout mouse line with no detectable HOXC10 in mature adipocytes revealed spontaneous subcutaneous WAT browning, increased expression of genes involved in browning, increased basal rectal temperature, enhanced cold tolerance, and improved glucose homeostasis. These phenotypes were further exacerbated by exposure to cold or a ß-adrenergic stimulant. Mechanistically, cold and ß-adrenergic exposure led to reduced HOXC10 protein level without affecting its mRNA level. Cold exposure induced cAMP-dependent protein kinase-dependent proteasome-mediated degradation of HOXC10 in cultured adipocytes, and shotgun proteomics approach identified KCTD2, 5, and 17 as potential E3 ligases regulating HOXC10 proteasomal degradation. Collectively, these data demonstrate that HOXC10 is a gatekeeper of WAT identity, and targeting HOXC10 could be a plausible therapeutic strategy to unlock WAT thermogenic potentials.

Oligomers of the lipodystrophy protein seipin may co-ordinate GPAT3 and AGPAT2 enzymes to facilitate adipocyte differentiation.

Seipin deficiency causes severe congenital generalized lipodystrophy (CGL) and metabolic disease. However, how seipin regulates adipocyte development and function remains incompletely understood. We previously showed that seipin acts as a scaffold protein for AGPAT2, whose disruption also causes CGL. More recently, seipin has been reported to promote adipogenesis by directly inhibiting GPAT3, leading to the suggestion that GPAT inhibitors could offer novel treatments for CGL. Here we investigated the interactions between seipin, GPAT3 and AGPAT2. We reveal that seipin and GPAT3 associate via direct interaction and that seipin can simultaneously bind GPAT3 and AGPAT2. Inhibiting the expression of seipin, AGPAT2 or GPAT3 led to impaired induction of early markers of adipocyte differentiation in cultured cells. However, consistent with normal adipose mass in GPAT3-null mice, GPAT3 inhibition did not prevent the formation of mature adipocytes. Nonetheless, loss of GPAT3 in seipin-deficient preadipocytes exacerbated the failure of adipogenesis in these cells. Thus, our data indicate that GPAT3 plays a modest positive role in adipogenesis and argue against the potential of GPAT inhibitors to rescue white adipose tissue mass in CGL2. Overall, our study reveals novel mechanistic insights regarding the molecular pathogenesis of severe lipodystrophy caused by mutations in either seipin or AGPAT2.

Discovering metabolic disease gene interactions by correlated effects on cellular morphology.

OBJECTIVE: Impaired expansion of peripheral fat contributes to the pathogenesis of insulin resistance and Type 2 Diabetes (T2D). We aimed to identify novel disease-gene interactions during adipocyte differentiation. METHODS: Genes in disease-associated loci for T2D, adiposity and insulin resistance were ranked according to expression in human adipocytes. The top 125 genes were ablated in human pre-adipocytes via CRISPR/CAS9 and the resulting cellular phenotypes quantified during adipocyte differentiation with high-content microscopy and automated image analysis. Morphometric measurements were extracted from all images and used to construct morphologic profiles for each gene. RESULTS: Over 107 morphometric measurements were obtained. Clustering of the morphologic profiles accross all genes revealed a group of 14 genes characterized by decreased lipid accumulation, and enriched for known lipodystrophy genes. For two lipodystrophy genes, BSCL2 and AGPAT2, sub-clusters with PLIN1 and CEBPA identifed by morphological similarity were validated by independent experiments as novel protein-protein and gene regulatory interactions. CONCLUSIONS: A morphometric approach in adipocytes can resolve multiple cellular mechanisms for metabolic disease loci; this approach enables mechanistic interrogation of the hundreds of metabolic disease loci whose function still remains unknown.

Cost effectiveness analysis of using quantitative ultrasound as a selective pre-screen for bone densitometry.

It has been suggested that quantitative ultrasound (QUS) could be used as a selective population pre-screen, to maximise the cost effectiveness of referral for dual energy X-ray absorptiometry (DXA) assessment of bone mineral density (BMD). We set out to examine how such an approach might perform in the assessment of women who were referred by general practitioners for DXA via the open access service in Cardiff. In 115 women aged 40-80 (mean 69) years we used DXA to measure BMD at lumbar spine and hip, and QUS to measure broadband ultrasound attenuation (BUA) in the heel. A bottom-up approach was used to estimate the costs of DXA and QUS. We examined the cost effectiveness of using QUS as a pre-screen, only referring subjects for the more expensive DXA assessment if BUA were less than a pre-determined threshold. The unit costs of pencil-beam DXA and QUS were approximately 44 UK pounds and 16 UK pounds respectively. We identified a BUA threshold of 60 dB/MHz as the most cost effective, and calculated a sensitivity of 81% and specificity of 89% in identifying those subjects whom DXA assessment subsequently identified as having osteoporosis. At the BUA threshold of 60 dB/MHz, pre-screening saved 969 UK pounds at the expense of missing ten women with osteoporosis as diagnosed by DXA. Therefore the cost per additional woman with osteoporosis identified using DXA alone was only 97 UK pounds. QUS assessment does not appear to have a significant cost effective benefit as a pre-screen for DXA in the studied population. A QUS pre-screen would be cost effective only if this investigation could be performed at a substantially lower cost.

Seipin oligomers can interact directly with AGPAT2 and lipin 1, physically scaffolding critical regulators of adipogenesis.

OBJECTIVE: Disruption of the genes encoding either seipin or 1-acylglycerol-3-phosphate O-acyltransferase 2 (AGPAT2) causes severe congenital generalized lipodystrophy (CGL) in humans. However, the function of seipin in adipogenesis remains poorly defined. We demonstrated recently that seipin can bind the key adipogenic phosphatidic acid (PA) phosphatase lipin 1 and that seipin forms stable dodecamers. As AGPAT2 generates PA, the substrate for lipin 1, we investigated whether seipin might bind both enzymes of this lipid biosynthetic pathway, which is required for adipogenesis to occur. METHODS: We employed co-immunoprecipitation and immunofluorescence methods to determine whether seipin can interact with AGPAT2 and the consequences of this in developing adipocytes. Atomic force microscopy was used to determine whether these interactions involved direct association of the proteins and to define the molecular architecture of these complexes. RESULTS: Our data reveal that seipin can bind AGPAT2 during adipogenesis and that stabilizing this interaction during adipogenesis can increase the nuclear accumulation of PPARγ. Both AGPAT2 and lipin 1 can directly associate with seipin dodecamers, and a single seipin complex can simultaneously bind both AGPAT2 and lipin with a defined orientation. CONCLUSIONS: Our study provides the first direct molecular link between seipin and AGPAT2, two proteins whose disruption causes CGL. Moreover, it provides the first example of an interaction between seipin and another protein that causally influences a key aspect of adipogenesis. Together our data suggest that the critical role of seipin in adipogenesis may involve its capacity to juxtapose important regulators of this process in a multi-protein complex.

An investigation of the sex difference in the diuretic response to ethacrynic acid in mice.

1. Female mice of the Hough/Porton and Tuck/TO strains were found to be more sensitive than male mice to the diuretic effects of oral and intravenous doses of ethacrynic acid. 2. The sensitivity of Hough/Porton male mice to ethacrynic acid was increased after pretreatment with stilboestrol and the sensitivity of female Hough/Porton mice decreased after pretreatment with testosterone. 3. There were no significant sex differences in the diuretic response to frusemide, acetazolamide, aminophylline, bendrofluazide, and Su 15049A although a small, but significant, increase in the sensitivity of male Tuck/TO mice to triamterene was noted. 4. The sex difference in diuretic response to ethacrynic acid may be related to an effect of sex hormone balance on its metabolism or on the sensitivity of its renal receptor.

BTS 39542, a dihydrophthalazin-1-ylacetic acid with high efficacy diuretic activity.

1 BTS 39542, a novel dihydrophthalazin-1-ylacetic acid, has high efficacy diuretic activity in mice, rats, rabbits and dogs. It is twice as potent as frusemide in mice and dogs, ten times as potent in rats and twenty times as potent in rabbits. 2 BTS 39542, like frusemide, exerts its major effects in the loop of Henle and increases renal blood flow but does not affect glomerular filtration rate in dogs. 3 The ratio of the excretion of the major cations (sodium plus potassium) to that of the major anion (chloride) after either BTS 39542 or frusemide varied with species. In rats and rabbits the ratio was approximately unity but in mice and dogs the ratio consistently exceeded unity. 4 A method for evaluating diuretics based on potency and relative potassium excretion is described.

Cardiovascular effects of the novel arteriovenous dilator agent, flosequinan in conscious dogs and cats.

1. Flosequinan (BTS 49 465, 7-fluoro-1-methyl-3-methylsulphinyl-4-quinolone) a novel arteriovenous dilator agent was orally effective in conscious renal hypertensive dogs and normotensive cats. The hypotensive potency of flosequinan was approximately ten times less than that of hydralazine in renal hypertensive dogs, 10 mg kg-1 and 20 mg kg-1 flosequinan causing similar falls in mean blood pressure to 1 mg kg-1 and 3 mg kg-1 hydralazine respectively. In normotensive cats, 5 mg kg-1 flosequinan caused similar falls to 0.5 and 1.0 mg kg-1 hydralazine. The onset of hypotensive effect after flosequinan appeared to be slightly slower than after hydralazine in the dog and slightly faster than hydralazine in the cat. 2. The degree of tachycardia and increase in plasma renin activity (PRA) for equivalent falls in mean blood pressure in both species was significantly less for flosequinan than for hydralazine (P less than 0.05). 3. In normotensive dogs, flosequinan, 10 and 20 mg kg-1 orally, caused a small but non-significant increase in sodium and chloride excretion and had little effect on urine volume whereas hydralazine, 1 and 3 mg kg-1 orally, caused a marked retention of sodium and chloride ions and a reduction in urine volume (P less than 0.01). 4. Neither flosequinan, 10 mg kg-1 orally, nor hydralazine 1 mg kg-1 orally, affected either glomerular filtration rate measured as creatinine clearance or effective renal plasma flow measured as p-aminohippuric acid clearance in normotensive dogs. 5. The lesser degree of tachycardia and increase in plasma renin activity together with a lack of sodium retaining activity associated with flosequinan suggest that this agent may have potential advantages over existing therapy as an antihypertensive in man.

Differential inotropic effects of flosequinan in ventricular muscle from normal ferrets versus patients with end-stage heart failure.

1. In right ventricular papillary muscles from control ferrets, flosequinan (10(-7)-10(-4) M) produced a concentration-dependent positive inotropic effect (10(-5) M = 153 +/- 24, 10(-4) M = 198 +/- 44% increase in isometric tension; control tension = 100%; n = 11) associated with a corresponding increase in amplitude of the intracellular Ca2+ ([Ca2+]i) transient recorded with aequorin (10(-5) M = 133 +/- 11, 10(-4) M = 187 +/- 36% increase in [Ca2+]i transient; n = 11). 2. The positive inotropic effect of flosequinan in control ferret ventricular muscle was neither blocked by propranolol (6 x 10(-7) M), nor associated with the abbreviation of the [Ca2+]i transient and contraction that is typical of catecholamines. 3. Neither flosequinan (n = 12) nor BTS 53 554, its sulphone metabolite (n = 6) produced a positive inotropic effect or altered the time course of contraction in myocardium from the hearts of patients with end-stage failure. 4. In contrast to milrinone, which produces a positive inotropic effect via phosphodiesterase inhibition, the unresponsiveness of myopathic human myocardium to flosequinan was not restored after intracellular adenosine 3':5'-cyclic monophosphate (cyclic AMP) levels were increased by prior treatment with forskolin (n = 13). 5. Taken together, these data indicate that flosequinan has a direct positive inotropic effect that is Ca(2+)-dependent, but independent of changes in intracellular cyclic AMP concentrations. 6. The positive inotropic effect may be species-dependent or altered by the presence of hypertrophy and/or heart failure. However, when used therapeutically in patients with severe heart failure, our data suggest that flosequinan should not adversely affect myocardial oxygen consumption through direct or catecholamine-mediated actions on the heart.

European reference values for Doppler indices of left ventricular diastolic filling.

The European Study Group on diastolic heart failure requires objective evidence of abnormal left ventricular diastolic function to establish the diagnosis of diastolic heart failure, which is common in older people. Reference values for Doppler indices of transmitral flow, used to assess left ventricular diastolic function, have not been reported for people 70 years and over in Europe. The aim of this study was to establish reference values for these Doppler indices of transmitral flow in older people. A random sample of 355 subjects aged 70 and over, living in the community underwent clinical assessment and echocardiography. Asymptomatic subjects with no cardiovascular disease and cardiovascular risk factors were identified. Measurements of five commonly used Doppler indices of transmitral flow from these subjects were obtained and reference range expressed as mean+/-2 standard deviations and as percentiles. We have therefore generated reference Doppler values of transmitral flow for people aged over 70 in a British population.

Relationship between inotropic activity and phosphodiesterase inhibition for flosequinan and milrinone.

The goal of this study was to assess the relationship between the positive inotropic response to high concentrations of the vasodilators flosequinan and BTS 53 554 (the sulfone metabolite of flosequinan) and the effect of both compounds on different forms of cyclic nucleotide phosphodiesterase. In addition, the relationship between inotropic activity and phosphodiesterase inhibition for the cardiotonic milrinone was also evaluated. All three agents exerted a positive inotropic effect on human cardiac muscle fibers. The concentration of milrinone required to increase cardiac contractility was comparable to the concentration required to inhibit the milrinone-sensitive subclass of cyclic AMP-specific phosphodiesterase (type III phosphodiesterase). However, no such relationship was observed for flosequinan and BTS 53 554. These results suggest that the cardiac response to high concentrations of flosequinan and BTS 53 554 is not mediated by inhibition of type III phosphodiesterase.

Effects of various diuretic agents in the mouse.

Diuretic effects of seven orally-acting diuretic agents have been examined in the mouse. The following compounds, examples of various types of orally active compound available, produced their characteristic diuretic effects: bendrofluazide, frusemide, ethacrynic acid, acetazolamide, triamterene, aminophylline and Su 15049A. The diuretic effects of the various agents were demonstrated under both water and saline-loading conditions. After allowing for differences in baseline sodium excretion, all diuretics except acetazolamide caused a further enhancement of sodium excretion after saline-loading compared with water-loading tests. The mouse possesses several advantages over the more commonly used rat since the range of diuretic responsiveness is greater. These results suggest that the mouse is a suitable species for diuretic testing.

Cost effectiveness analysis of BMD referral for DXA using ultrasound as a selective pre-screen in a group of women with low trauma Colles' fractures.

Measurements of bone density (BMD) are central to the World Health Organisation (WHO) approach to the definition of osteoporosis. Dual energy X- ray absorptiometry (DXA) remains the gold standard technique for measuring the bone mineral density (BMD) but Quantitative Ultrasound (QUS) is an attractive alternative method of bone assessment because it is easy to use and relatively inexpensive. It has been suggested that QUS could be used as a selective population pre-screen, to maximise the cost effectiveness of referral for DXA assessment of BMD. We set out to examine how such an approach might perform in the assessment of women with low trauma Colles' fracture. In 46 women aged 50-80 (mean 67) years we used DXA to measure BMD at lumbar spine and hip, and heel bone ultrasound to measure Broad Band Attenuation (BUA) and Velocity of Sound (VOS). We calculated local costs of pounds sterling 45 for DXA and pounds sterling 15 for QUS. We identified a BUA threshold of 60 dB/MHz as most cost effective as pre-screen, and calculated a sensitivity of 93% and specificity of 84% in identifying those subjects who were subsequently identified as having osteoporosis by DXA. DXA assessment of all patients had a cost of pounds sterling 77 per osteoporotic subject identified. We examined the cost-effectiveness of using QUS as a pre-screen, only referring subjects for more expensive DXA assessment if BUA was less than 60 dB/MHz. However this approach had no advantage, still costing pounds sterling 78 per osteoporotic subject identified. QUS assessment does not appear cost-effective as a pre-screen for DXA, even in this high risk group of women with low trauma Colles' fracture. A QUS pre-screen would only be cost-effective if the scan could be performed at a substantially lower cost.

Analyzing the functions and structure of the human lipodystrophy protein seipin.

Disruption of the gene BSCL2, which encodes the protein seipin, causes severe generalized lipodystrophy in humans with a near complete absence of adipose tissue. Moreover, cell culture studies have demonstrated that seipin plays a critical cell-autonomous role in adipocyte differentiation. These observations reveal seipin as a critical regulator of human adipose tissue development; however, until recently very little has been known about the potential molecular functions of this intriguing protein. Despite significant recent interest in the function of seipin, our understanding of its molecular role(s) remains limited. The topology of seipin and lack of evidence for any enzymatic domains or activity indicate that it may act principally as a scaffold for other proteins or play a structural role in altering membrane curvature and/or budding. Work in this area has been hampered by several factors, including the lack of homology that might imply testable functions, the poor availability of antibodies to the endogenous protein and the observation that this hydrophobic ER membrane-resident protein is difficult to analyze by standard Western blotting techniques. Here we summarize some of the techniques we have applied to investigate the association of seipin with a recently identified binding partner, lipin 1. In addition, we describe the use of atomic force microscopy (AFM) to image oligomers of the seipin protein. We believe that AFM will offer a valuable tool to examine the association of candidate binding proteins with the seipin oligomer.

The human lipodystrophy protein seipin is an ER membrane adaptor for the adipogenic PA phosphatase lipin 1.

Disruption of the gene BSCL2 causes a severe, generalised lipodystrophy, demonstrating the critical role of its protein product, seipin, in human adipose tissue development. Seipin is essential for adipocyte differentiation, whilst the study of seipin in non-adipose cells has suggested a role in lipid droplet formation. However, its precise molecular function remains poorly understood. Here we demonstrate that seipin can inducibly bind lipin 1, a phosphatidic acid (PA) phosphatase important for lipid synthesis and adipogenesis. Knockdown of seipin during early adipogenesis decreases the association of lipin 1 with membranes and increases the accumulation of its substrate PA. Conversely, PA levels are reduced in differentiating cells by overexpression of wild-type seipin but not by expression of a mutated seipin that is unable to bind lipin 1. Together our data identify lipin as the first example of a seipin-interacting protein and reveals a novel molecular function for seipin in developing adipocytes.